Design of antibody variable fragments with reduced reactivity to preexisting anti-drug antibodies

Upon reformatting of an antibody to single-chain variable fragment format, a region in the former variable/constant domain interface of the heavy chain becomes accessible for preexisting (PE) anti-drug antibody (ADA) binding. The region exposed because of this reformatting contains a previously hidden hydrophobic patch. In this study, mutations are introduced in this region to reduce PE ADA reactivity and concomitantly reduce the hydrophobic patch. To enhance our understanding of the importance of individual residues in this region with respect to PE ADA reactivity, a total of 50 molecules for each of two antibodies against different tumor-associated antigens were designed, produced, and characterized by an arsenal of biophysical methods. The aim was to identify suitable mutations that reduce, or completely eliminate, PE ADA reactivity to variable fragments, without compromising biophysical and pharmacodynamic properties. Computational methods were used to pinpoint key residues to mutate and to evaluate designed molecules in silico, in order to reduce the number of molecules to produce and characterize experimentally. Mutation of two threonine residues, Thr101 and Thr146 in the variable heavy domain, proved to be critical to eliminate PE ADA reactivity. This may have important implications in optimizing early drug development for antibody fragment-based therapeutics.

Automated summary

Upon reformatting antibody to single-chain variable fragment format, a previously hidden hydrophobic patch in the variable/constant domain interface becomes accessible for preexisting anti-drug antibody binding. To reduce the reactivity of preexisting antibodies and the exposed hydrophobic patch, mutations were introduced in this region. Through computational methods and experimental characterization, mutation of two threonine residues, Thr101 and Thr146, was found to be critical in eliminating preexisting antibody reactivity, which could have important implications in optimizing early drug development for antibody fragment-based therapeutics.